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Mohammad Shahidullah, Amritlal Mandal, Nicholas A Delamere; Hemichannel opening in the lens epithelium involves Rho kinase. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5739. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We have shown earlier that hyposmotic stress opens hemichannels (HCs) in the lens epithelium. The HCs release ATP that subsequently activates receptors, Src family kinases (SFKs) and stimulates Na,K-ATPase activity. Here we show evidence that the HC opening mechanism involves Rho kinase (ROCK).
Intact porcine lenses were exposed to isosmotic (300mOsm) or hyposmotic solution (200 mOsm) containing propidium iodide (PI) which was added to probe for HC opening. Propidium iodide (PI) uptake was quantified by fluorimetry. ATP release from the lens epithelium was measured by luciferin-luciferase bioluminescence. Calcium entry into cultured lens epithelium was measured using the calcium-sensitive dye Fura-2.
Hyposmotic solution caused a robust increase in PI uptake by the epithelium of intact lenses. When lenses were exposed to hyposmotic solution for 30 min, PI fluorescence in the epithelium was 9.6±0.9 compared to 2.7±2.2 AU/mg protein (n=5) in the epithelium of control (isosmotic) lenses. When lenses were exposed to hyposmotic solution in the presence of a selective ROCK inhibitor Y-27632 (20 μM), the magnitude of PI uptake was significantly reduced to 4.8±0.8. PI uptake did not change significantly in the epithelium of lenses exposed to Y-27632 alone (2.8±0.8). Y-27632 reduced ATP release into the bathing solution when lens epithelum was exposed to hyposmotic solution: ATP bioluminescence in the medium was 86±19 compared to 808±150 (n=8) (arbitrary units) in the presence vs absence of Y-27632. Consistent with HC-mediated calcium entry, hyposmotic solution caused a sustained calcium increase in cultured epithelium. The calcium increase was abolished by Y-27632. Hyposmotic solution also was found to cause rapid phosphorylation of myosin light chain2 (MLC2) as determined by quantitative western blot analysis.
The ability of Y-27632 to inhibit PI uptake, ATP release and calcium entry all are consistent with suppression of HC opening. The findings point to a critical role for ROCK in the mechanism of epithelial HC opening when lenses are subjected to hyposmotic stress.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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